Angular difference in human coronary artery governs endothelial cell structure and function

Blood vessel branch points exhibiting oscillatory/turbulent flow and lower wall shear stress (WSS) are the primary sites of atherosclerosis development. Vascular endothelial functions are essentially dependent on these tangible biomechanical forces including WSS. Herein, we explored the influence of blood vessel bifurcation angles on hemodynamic alterations and associated changes in endothelial function. We generated computer-aided design of a branched human coronary artery followed by 3D printing such designs with different bifurcation angles. Through computational fluid dynamics analysis, we observed that a larger branching angle generated more complex turbulent/oscillatory hemodynamics to impart minimum WSS at branching points. Through the detection of biochemical markers, we recorded significant alteration in eNOS, ICAM1, and monocyte attachment in EC grown in microchannel having 60o vessel branching angle which correlated with the lower WSS. The present study highlights the importance of blood vessel branching angle as one of the crucial determining factors in governing atherogenic-endothelial dysfunction.

2. The conclusions of this study are heavily based upon immunofluorescence data from different regions of the microchannel. It would be more convincing if the authors could provide immunofluorescence images of the entire microchannel with the resolution to visualize the fluorescence intensity changes across regions with different flow patterns. 3. The title states "Angular difference……governs endothelial cells structure and function……". However, only two markers were used to assess EC states in different flow regions. No functional assays were performed to confirm the atheroprone phenotypes. 4. Flow-induced changes in gene expression and EC phenotypes in vitro are highly dependent on the duration of exposure to flow. The authors should justify why a 4-hour flow was selected for the study. Could the changes in eNOS/ICAM1 expression and EC morphology be transient responses?
5. According to Supplemental Fig. 1d, no pulse dampener was used in conjunction with the peristaltic pump. Could the liquid pulses lead to more complicated D-flow patterns in the microchannels and modulate EC phenotypes?
Reviewer #2 (Remarks to the Author): The study is about an in-vitro and in-silico model of a coronary artery bifurcation and investigates different bifurcation angles on the wall shear stress and accumulation of plaques. While the study's goal is well-intentioned and aimed in the right direction, that is to translate computational techniques to in vitro and eventually to the clinic, there are major limitations that need to be addressed. This paper can be recommended for publication in the case authors make major changes to the manuscript. 1. It is not clear what is the major contribution of your work specially in model development and how the current study is related to the medical needs. 2. Based on the manuscript it's hard to identify the link between in silico model and in vitro study. Is there any in silico data used for the in silico model? What kind of framework that you developed? Authors used the native ANSYS solver and it's difficult to see any contribution as development in computational modelling. 3. Despite the use of a 3D CFD simulation of a coronary artery bifurcation, the boundary conditions employed for the study are quite simple and unrealistic. The authors should have used the pulsatile flow rate at the inlet and a 3 element Windkessel model representing resistance, compliances etc. at the outlets. Then they were able to compute time average wall shear stress and oscillatory shear index which based can show a better picture of the low shear regions and the recalculation zones for governing atherogenic-endothelial dysfunction and accumulation of plaques. 4. As authors on page 9, lines 284-256 stated that "Even though the flow is laminar in the entrance region, however, it may turn into low-Re turbulent 282 in the bifurcation region. SST k-ω model31 was considered to achieve accurate results when fluid is in low-Re turbulence and the viscous-sublayer region." However, the low-Re turbulent models are used for the turbulent flows to capture the low-Reynolds region close to the wall where viscous effects are dominant. Based on the inlet velocity and diameter on a rough calculation the Reynolds number at the entrance is approximately 90 and by checking the maximum velocity the bifurcation with steady-state condition Reynolds number always remains very low and the choice of using the turbulent model is not correct which gives unrealistic results for the WSS calculations. 5. Despite the detailed model of in vitro, the current study adds only incremental value to the plethora of studies that are already present in the literature. For example in page 7 line 233-234 "Therefore, it would be alright to conclude that blood vessels with bifurcation angles between 50 and 60° are at a higher risk of accumulating plaques. " as a summary of your work which already can be found based on literature. 6. The explanation for in vitro setup and how this setup is different from previous studies, any novelty? 7. The choice of using non-Newtonian fluid for their in silico model is realistic. However, the medium that was used in the experiments has to be examined with a rheometer and its characteristics need to be consistent with the modified-Casson model that is used in silico. . However, with relatively poor representative images and without appropriate discussion of the data in the results section, the D-Flow patterns remained undiscussed in the previously submitted manuscript. In the revised version of the manuscript, we elaborated on this based on our in silico and experimental data (Page 6, Paragraph 2, and lines 13-23). Furthermore, we have performed new analysis and experiments to address the referees concern. We have now reported the velocity vectors using better and high resolution zoomed images (Figure 2a-f) and deliberated about the D-Flow patterns on Page 6, Paragraph 2, and lines 13-23. In addition, using real-time videography facility of an inverted microscope adapted with a video recording camera, we recorded the movement of magnetic beads through the microchannels containing 30, 60 and 80 degree bifurcation angles (Supplemental videos 1-7). Such videos clearly indicated slower movement with bead taking dips within the bifurcation region of microchannels containing 60 and 80 degree branching angles. The Referee's points concerning the spatial determination of the D-Flow regions and the consistency of immunofluorescence imaging have been addressed further. As apparent from the representation in Figure 2o, based on the reference area as indicated by circles in WSS contour and tile scan images, the spatial accuracy and consistency have been maintained throughout the study where immunofluorescence imaging was undertaken. Moreover, we have performed tile scan imaging (that provides a broader view of the entire "Y" shaped structure) of both eNOS ( Figure 3d) and ICAM1 ( Figure 4d) staining of microchannels containing 30, 60 and 80 degree branching angles, and we observed parallel findings to that of the higher magnification images. I hope these new experiments, analysis and elaborate discussion address referee's this comment. Fig. 1h-i (Figure 2o) was used to determine the precise location of the D-Flow regions in the microchannels. We have now included a microscopic image of the microchannel (Figure 2o) to better visualize the areas that were selected for imaging. Based on the respective WSS data, all the microscopic images were taken at similar locations in the microchannels with different angles (just after the outer-wall curvature and below the inner branch point) as depicted in Figure 2o.

Are the results presented in
To further validate the D-Flow effect and its location, we setup our flow module under an inverted microscope; circulated magnetic beads suspended in PBS, and recorded the videos at various locations (where microscopic imaging was performed throughout this study) (Supplemental Videos 1-7). As described earlier, such videos clearly indicated slower movement with bead taking dips within the bifurcation region of microchannels containing 60 and 80 degree branching angles.
2. The conclusions of this study are heavily based upon immunofluorescence data from different regions of the microchannel. It would be more convincing if the authors could provide immunofluorescence images of the entire microchannel with the resolution to visualize the fluorescence intensity changes across regions with different flow patterns.

Response:
As suggested by the Referee, we captured the images of the microchannel at the lowest available magnification (5x), however, we failed to capture the entire microchannels. We therefore took 5x images of the regions precisely used for 20x imaging and fluorescence intensity analysis, and have included them to complement the respective 20x images (Supplemental Figure  4). Furthermore, to visualize a greater part of the microchannels along with the vessel branching points, we performed tile scan imaging using the confocal facility available at BITS Pilani, Pilani Campus. The tile scan images presented in Figure 3d (eNOS) and Figure 4d (ICAM1) of microchannels containing 30, 60 and 80 degree branching angles, we observed parallel findings to that observed with the higher magnification images.

Response:
We thank the Referee for bringing this point. The association between monocytes and atherosclerotic lesions, both in animal models and in humans, has long been recognized 1,2 . We therefore performed monocyte adhesion assay to confirm the atheroprone phenotypes of the EC at vessel branch points. This new functional data is now included in Figure 5. This data clearly revealed highest attachment of monocyte to endothelial monolayer at D-variable regions of bifurcation in microchannel containing 60 0 branching points, especially in comparison to microchannel containing 30 0 branching points. In addition, we have further performed more structural analysis of EC by staining the cells with cell-cell contact marker CD144 ( Figure 6) and by staining the cell F-actin using phalloidin-TRITC ( Figure 6, Supplemental Figure 5a). Furthermore, we also performed a comprehensive structural analysis by performing additional experiments to capture SEM images of the cells to ascertain structural and surface level changes in the cells in response to differential bifurcation of the microchannels (Supplemental Figure 5a). All these data revealed significant structural deformations of the cells exposed to differential flow conditions at D-variable regions of the mcirochannels containing 60 and 80 degree branching angles.

Flow-induced changes in gene expression and EC phenotypes in vitro are highly dependent on the duration of exposure to flow. The authors should justify why a 4-hour flow was selected for the study. Could the changes in eNOS/ICAM1 expression and EC morphology be transient responses?
Response: We like to thank the referee for asking a very pertinent question. Evidence suggests that flow dependent changes in eNOS/ICAM1 expression and EC morphology are not transient. Previous findings have shown phenotypic changes in EC exposed to D-Flow for as low as 1 hour 3 . Similarly, our precursory publication shows how a 4 hours D-Flow exposure epigenetically regulates endothelial inflammation and apoptosis 4 . During that study we performed a time dependent D-Flow exposure (4, 8, 12, and 24 hours) experiment, using the microchannels, and assessed eNOS and ICAM1 protein levels. The changes in the expression levels were similar and comparable for all the time points. Because we observed a robust induction of endothelial inflammatory phenotype (reduction in eNOS and increase in ICAM1) within 4 hours of fluid flow exposure, we went ahead and carried out all our microchannels' experiments with a 4 hours D-Flow exposure set up. Fig. 1d, no pulse dampener was used in conjunction with the peristaltic pump. Could the liquid pulses lead to more complicated D-flow patterns in the microchannels and modulate EC phenotypes?

Response:
We again like to appreciate the point raised by the Referee. The absence of a pulse dampener surely affects the constant flow rate. However, increasing rollers does tend to decrease the amplitude of the fluid pulsing at the outlet by increasing the frequency of the pulsed flow. The peristaltic pump that we employed had six rollers, thereby maintaining a constant-like flow rate. Moreover, our new simulation studies with pulsatile flow shows circulatory flow patterns at the D-Flow regions, while the WSS follows a similar trend -with increasing angles of bifurcationswhen compared to constant/steady-state flow (Figure 1g). Thus, our current microfluidics setup offers a more realistic in vivo-like fluid flow conditions.

Response to the queries of Referee #2
The study is about an in-vitro and in-silico model of a coronary artery bifurcation and investigates different bifurcation angles on the wall shear stress and accumulation of plaques. While the study's goal is well-intentioned and aimed in the right direction, that is to translate computational techniques to in vitro and eventually to the clinic, there are major limitations that need to be addressed. This paper can be recommended for publication in the case authors make major changes to the manuscript.
We like to thank the Referee for positive review of our study and for suggesting a comprehensive plan for revising the manuscript that we believe significantly elevated to quality of the study.

It is not clear what is the major contribution of your work specially in model development and how the current study is related to the medical needs.
Response: Thank you for bringing this point. The in vitro platform employed in the study mimics in vivo-like fluid flow conditions. The use of 3D-printed human coronary artery for casting microchannels, proffer natural vessel curvature and disturbed in vivo branching point-like flow patterns. The existing eNOS and ICAM1 protein expression data along with the newly included monocyte adhesion data ( Figure 5) confirms that our model is capable of inducing pro-atherogenic endothelial phenotypes. In our previous publication -employing this microchannels' model -we circulated a pharmacological inhibitor at appropriate concentrations and reported that the drug successfully inhibited EC phenotypic alterations 4 . We have further elaborated on the significance of our model and the clinical relevance of this study under the results and discussion section at Page 9, Paragraph 2, and lines 18-27. In addition, in this paper, we study the effect of angular bifurcation on the minimum WSS using various models such as steady state, pulsatile-rigid arterial wall, and Pulsatile with Fluid-Structure-Interaction between arterial wall and fluid. We believe the model development is comprehensive, considering all the real flow scenarios. In the literature, we found studies which only consider one or the other assumption. The crux of the paper is to demonstrate and employ CFD to find the correlation between the WSS and biological response in the in vitro experimental set up. The framework developed includes the CAD-based geometries for both in silico and in vitro models, which would give accurate predictions of WSS and other hemodynamic descriptors.
We believe that the paper will be of value to the medical field as well since the paper (a) demonstrates a correlation between WSS and endothelial response, and (b) usage of CFD to study the flow patterns and predict the biological response.
2. Based on the manuscript it's hard to identify the link between in silico model and in vitro study. Is there any in silico data used for the in silico model? What kind of framework that you developed? Authors used the native ANSYS solver and it's difficult to see any contribution as development in computational modelling.

Response:
We appreciate the comment provided by the referee. In the present study, our main aim was to use in silico CFD analysis with ANSYS solver and correlate such data with biological response achieved through in vitro cell culture based experiments to establish a correlation between the effect of angular bifurcation and the minimum WSS to that of the biological responses in cellular level. To the best of our knowledge, to date no study amalgamated these two analysis and observations together to achieve the goal of the present study; correlation of CFD based analysis with biological response in cellular level through cell culture based experimentation. CFD analysis performed in the current study allowed us to identify the regions in the bifurcation area in context to deviation in WSS with changes in branching angel and further asses the biological response based on such changes in fluid dynamics and WSS on those areas. As for the model development, with new set of in silico analysis, we undertook a comprehensive effort to include all parameters, reducing the assumptions to the minimum to make it more realistic. In the revised manuscript through new sets of CFD analysis, we now report CFD analysis of steady-state simulations, pulsatile with the rigid wall, and pulsatile with the fluid-structure interaction model to achieve an exhaustive analysis to ascertain the variation in WSS and velocity streamlines between these specified models. Moreover, through such analysis, we affirm that indeed WSS at bifurcation angel remain to be proportionally altering based on the changes in the branching angels of the microchannels. In specific, WSS at the branching point of microchannel with 60 0 angel of bifurcation remained to be the lowest between all these newly reported analysis using different boundary conditions (Figure 1g).  (Figure 1g). All these new data have been included and discussed in the main text of the manuscript to address the referee's comment.

4.
As authors on page 9, lines 284-256 stated that "Even though the flow is laminar in the entrance region, however, it may turn into low-Re turbulent 282 in the bifurcation region. SST k-ω model31 was considered to achieve accurate results when fluid is in low-Re turbulence and the viscous-sublayer region." However, the low-Re turbulent models are used for the turbulent flows to capture the low-Reynolds region close to the wall where viscous effects are dominant. Based on the inlet velocity and diameter on a rough calculation the Reynolds number at the entrance is approximately 90 and by checking the maximum velocity the bifurcation with steady-state condition Reynolds number always remains very low and the choice of using the turbulent model is not correct which gives unrealistic results for the WSS calculations.

Response:
In the new version of the CFD studies, our inlet boundary conditions were pulsatile, which showed that there is recirculation even with a low Reynolds number. SST k-W model has been used in the literature for various studies with pulsatile flow.

Despite the detailed model of in vitro, the current study adds only incremental value to
the plethora of studies that are already present in the literature. For example in page 7 line 233-234 "Therefore, it would be alright to conclude that blood vessels with bifurcation angles between 50 and 60° are at a higher risk of accumulating plaques. " as a summary of your work which already can be found based on literature.

Response:
As pointed by the Referee, we have modified the manuscript accordingly at Page 9, Paragraph 2, last part of the discussion. The main novelty of the work is to use CFD tools to correlate the relationship between the low shear stress region in the bifurcation and find the biological response in the same region for validation. We have used CAD files for printing the in vivo model and the same CAD geometry is used for the CFD simulations, which enabled us to build a strong correlation study between the CFD analysis and biological response in endothelial cell culture models. To the best of our knowledge, to date no study amalgamated these two analysis and observations together to achieve the goal of the present study; correlation of CFD based analysis with biological response in cellular level through cell culture based experimentation.

The explanation for in vitro setup and how this setup is different from previous studies, any novelty?
Response: It has now been discussed about under the results and discussion section at Page 9, Paragraph 2, last part of the discussion. We believe the main novelty of the study lies in the model system itself wherein we generated a computational model of a branched right human coronary artery for both 3D-printing followed by biological experimentation and CFD analysis using the same human coronary artery geometry. To date no studies, exist where they used such model system to assess the biological response based angular differences in branching point. More importantly, as stated earlier, to the best of our knowledge, to date no study amalgamated these two analysis and observations together to achieve the goal of the present study; correlation of CFD based analysis with biological response in cellular level through cell culture based experimentation.
7. The choice of using non-Newtonian fluid for their in silico model is realistic. However, the medium that was used in the experiments has to be examined with a rheometer and its characteristics need to be consistent with the modified-Casson model that is used in silico.

Response:
The culture medium used for in vitro fluid flow exposure contained 10% fetal bovine serum; the rheological analysis of the same gave a viscosity of 2.027 mPa.s, i.e, somewhere in between that of blood (3 mPa.s) and water (1 mPa.s). Furthermore, we found the medium circulated through the microchannels to have shear-thinning properties similar to that of a Casson fluid (Figure given below).

It's highly recommended that authors add a Limitation section in the manuscript.
Response: As suggested by the Referee, we have added few lines stating the limitations of the study at Page 9, Paragraph 2, lines 10-18.